Najib M. El-Sayed - US grants

African trypanosomes are protozoan parasites that cause a fatal disease called sleeping sickness in humans and ngana in domestic livestock. During the past two decades, an estimated 238 million dollars has been spent worldwide on basic research on African trypanosomes. This research has resulted in many advances in our understanding of not only these organisms, but also a number of previously unknown biological phenomena, some of which have been subsequently found to occur in cells of other organisms including mammals. Yet, these substantial research efforts have not led to a better way to manage or control this devastating disease. Recently, progress on the human genome project, and the determination of the yeast genomic sequence, have unambiguously demonstrated the value of using genomic sequences as a foundation for designing future research efforts. Our long term goals is to sequence the genome of Trypanosoma brucei. This proposal represents the first step towards that goal, and we propose the following strategy consisting of two phases. In the first phase, we will generate about 20 Mb of discontinuous single-pass sequence (74 percent of the 27 Mb non-minichromosomal genome). This will be implemented by end-sequencing of 10,000 already-existing clones (currently being used to assemble the physical map of T. brucei isolate TREU 927/4DNA), 5000 BAC clones (currently being made by Sara Melville, Cambridge Univ., UK) and 5000 small insert plasmid clones of randomly- sheared DNA (to be constructed by TIGR). Telomere-proximal sequences containing the telomere-linked vsgs will be identified in clones of the sheared genomic library. The purpose of this first phase is to enhance early gene discovery and to provide markers that will be important for construction of a high-resolution sequence-ready map. During the second phase, 10 Mb of contiguous T. brucei DNA will be sequenced using a chromosome by chromosome approach. This project will provide invaluable information and benefits at many levels, including (1) identification of genes involved in basic functions of the eukaryotic cells, (2) easy, inexpensive and fast cloning of genes encoding proteins being actively studied in laboratories around the world, (3) immediate access to genes and their products from functional/structural studies, (4) prediction of metabolic-pathways on the basis of candidate genes, and (5) identification of parasite-specific gene projects by comparison with other genomes.

Trypanosoma cruzi is a protozoan parasite that causes a fatal illness called Chagas' disease or American typanosomiasis in humans and many nonhuman mammalian species. The World Health Organization has estimated that 18 million people are infected with T. cruzi and that 50,000 patients die each year of the disease. The basic research on T. cruzi over the past two decades has resulted in many advances in our understanding of these organisms, yet these substantial research efforts have not led to a better way to manage or control this devastating disease. Recently, progress on the human genome project, and the determination of the yeast genomic sequence, have unambiguously demonstrated the value of using genomic sequences as a foundation for designing future research efforts. Our long term goal is to sequence the genome of T. cruzi. This proposal, in conjunction with similar proposed efforts at the Seattle Biomedical Research Institute and Uppsala University, represents a large step towards that goal. We will employ a sequencing strategy consisting of two phases. The purpose of the first phase is to enhance early gene discovery and to provide markers that will be important for construction of a high-resolution sequence-ready map. In this phase we will generate approximately 10 Mb of discontinuous single-pass sequence (23 percent of the 43.5 Mb genome). This will be implemented by end-sequencing 5,000 BAC (Bacterial Artificial Chromosome) clones from an already existing library and another 5,000 BAC clones of randomly-sheared DNA (from a library to be constructed in collaboration with Dr. Pieter deJong). Telomere-proximal sequences will be identified in clones of the sheared genonmic library. During the second phase 12 Mb of contiguous T. cruzi DNA will be sequenced using a chromosome by chromosome approach and a syntenic regions conserved between T. cruzi and a related parasite (Trypanosoma brucei) will be identified and analyzed. This project will provide invaluable information and benefits at many levels, including (1) identification of genes involved in basic functions of the eukaryotic cell, (2) easy, inexpensive and fast cloning of genes encoding proteins being actively studied in laboratories around the world, (3) immediate access to genes and their products for functional/structural studies, (4) prediction of metabolic pathways on the basis of candidate genes, (4) identification of parasite-specific gene products by comparison with other genomes, and (5) a framework for future experiments aimed at comparative and integrative mapping of various trypanosomatid genomes.

DESCRIPTION (Investigator's Abstract): Schistosoma mansoni is a trematode parasite that causes schistosomiasis. After malaria, schistosomiasis is the most prevalent tropical disease and leading cause of severe morbidity in developing nations. The disease is endemic in 74 developing countries, with estimates of 600 million people at risk and over 200 million infected. The schistosome genome is approximately 270 Mb, organized into 8 chromosome pairs. Under the auspices of the WHO, an international consortium was established in the 90's to encourage and coordinate genomic research on schistosomes. Its overall aim is to gain knowledge about the genome of these parasites in order to further understanding of their biology, mechanisms of drug resistance and antigenic variation that determine escape from the host's immune system. One of the main objectives of this program is the discovery and characterization of new genes of S. mansoni and in an attempt to search for new targets for drugs and vaccine development. As a preliminary step to achieving this goal, Dr. Cummings and her colleagues at TIGR will sequence 7 Mb of discontinuous DNA sequence using a BAC-end sequencing approach to obtain STS randomly distributed across the genome at about every 20 kb. Flourescent in situ hybridization (FISH) to mitotic metaphase chromosomes will be used to map the chromosomal locations of hundreds of these markers. The PI will also oversee sequencing and annotation of 5 Mb on contiguous DNA sequence from chromosome 3, ch3. A minimum BAC tiling path for a region near the centromere and long arm of ch3 is currently being constructed. For this purpose, terminal sequences and restriction patterns will be defined for each BAC clone, A series of 10 percent overlapping BACs will be selected and used to prepare BAC shotgun small insert sublibraries which will be sequenced to 8 fold coverage. The DNA sequence from this region will be assembled and following gap closure will be full annotated leading to the identification of perhaps hundreds of new schistosome genes which will revel new insights into schistosome chromosome and genome organization.

DESCRIPTION (provided by applicant): Summary Analysis of gene expression in pathogens and investigation of host-pathogen interactions are a critical step in fueling new drug and vaccine discovery projects. In this study, we focus on the protozoan parasite Leishmania major, the causative agent of human cutaneous leishmaniasis. While significant progress has been made over the past 20 years toward understanding the molecular and cellular basis for intracellular survival of Leishmania, we still lack information regarding the biology of intracellular amastigotes. In particular, little is known about gene expression in the parasite prior to and following phago-lysosomal fusion and the genes required to facilitate adaptation to, and survival within, the host cell. Furthermore, genes that are uniquely activated and expressed in the human macrophage, in the context the infection, have not been systematically determined. The recent emergence of next-generation sequencing technologies opens up unprecedented opportunities to gain an in-depth view of the transcriptome of L. major and the macrophage in the context of infection. The goal of this study is to obtain the first comprehensive transcriptome profiling data for the three main developmental stages of L. major (Aim 1) and to simultaneously analyze gene expression programs in the intracellular amastigote stage of L. major along with the trancriptional response of the infected human host cell, the macrophage (Aim 2). These studies should provide insight into host defense mechanisms initiated by macrophages and lead to a better understanding of what the macrophage sees on the parasite as it is being ingested. To further examine the interactions between the parasite and the human macrophage, we will identify a subset of important Leishmania-human protein-protein interaction (PPI) partners using well-established yeast two-hybrid screens (Aim 3). Very few experimental studies have investigated host-pathogen PPIs. Extending those to the intracellular parasite Leishmania will not only allow the identification PPIs that enable this parasite to infect its host cells, acquire nutrients and evade its immune defenses, but will also provide a more global functional view of pathogenesis in general. In summary, this study will provide the first massive-scale and simultaneous interrogation of the transcriptomes of an intracellular protozoan parasite and its macrophage host cell and will provide a first look at the infectome, the part of a host cell's genome and proteome that is important for infection by a pathogen as well as the part of the pathogen's genome/proteome that allows it to subvert the functions of some host cell receptors, signaling proteins and molecular machinery. .

DESCRIPTION (provided by applicant): Exploiting genome-scale approaches to identify pathogen-specific processes remains a promising strategy for the identification of novel targets for disease intervention. In the present study, we focus on the kinetoplastid protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas'disease. The currently available drugs to treat Chagas'disease are toxic and lack efficacy in the chronic stage of infection. The goal of this collaborative study is to exploit the use of Next-Generation Sequencing (NGS) technologies to obtain the first comprehensive gene expression profiling data for the four main developmental stages of T. cruzi (Aim 1) and to establish conditions that will permit the parallel analysis of gene expression programs in both intracellular parasites and their host cells (Aim 2). To achieve these goals, we will conduct high-resolution sequencing of the transcriptomes of T. cruzi CL Brener (the reference strain) and infected human cells (Aim2) by using an RNA-seq approach on the IlluminaZ platform. An extensive array of computation tools and experience in genome analysis will be applied to reconstruct full-length transcripts from the sequencing data and to map these to the T. cruzi and human genomes. We will define trans-splicing and polyadenylation sites and identify novel T. cruzi genes that were likely missed during the original annotation, thereby greatly enhancing current gene model structures and annotations. Using a suite of algorithms, mRNA abundance will be determined for every T. cruzi gene in each of four life cycle stages enabling us to identify co-expression patterns that correlate with the biology of the parasite. For the more complex scenario of deciphering parasite and host transcriptomes from infected cells, conditions will be optimized for T. cruzi infection of human vascular smooth muscle cells (VSMC) to ensure maximal coverage of parasite and host transcripts. Once this is established, we will conduct a limited time course of infection in VSMC to obtain a preliminary analysis of the dynamic nature of parasite and host cell gene expression programs in the context of infection. These data will provide the first glimpse of T. cruzi gene expression programs that are uniquely activated in the context of intracellular infection along with the transcriptional response of the human host cell. Results from this study, including enhanced annotations and expression profiling data, will be disseminated throughout existing public databases in the form of primary datasets and through Genbank and TritrypDB (www.tritrypdb.org), where the current genome data is maintained and curated. These studies will provide a solid framework for future functional and genomic studies in our own laboratories, as well as others in the broader Chagas's disease and intracellular parasitism fields. PUBLIC HEALTH RELEVANCE: Chagas'disease is a tropical parasitic disease that develops over a number of years in individuals that are chronically infected with the protozoan parasite, Trypanosoma cruzi. As a leading cause of heart failure in Latin America, Chagas'disease represents a public health problem of exceptional importance in endemic countries as well as an emerging immigrant health issue in the United States. The proposed study aims to use new DNA sequencing technologies to identify expressed genes in mammalian-infective T. cruzi stages on a genome-wide scale. Results from this work will provide novel insights into T. cruzi pathogenesis and guide efforts toward effective prevention or control of Chagas'disease.